8536d_ch05_105-136 8/22/02 2: 46 PM Page 108 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e 108 PART I1 Generation of B-Cell and T-Cell Response Tonegawa's Bombshell--Immunoglobulin myeloma cells), the V and C genes undergo rearrangement. Genes Rearrange In the embryo, the V and C genes are separated by a large DNA segment that contains a restriction-endonuclease site; evidence that separate genes encode the V and C regions of together and the intervening DNA Sequence is eliminated In 1976, S. Tonegawa and N. Hozumi found the first direct during differentiation, the V and C genes are brought closer mmunoglobulins and that the genes are rearranged in the The pioneering experiments of Tonegawa and Hozumi course of B-cell differentiation. This work changed the field employed a tedious and time-consuming procedure that has of immunology. In 1987, Tonegawa was awarded the Nobel since been replaced by the much more powerful approach of Prize for this work. Southern-blot analysis. This method, now universally used to Selecting DNA from embryonic cells and adult myeloma investigate the rearrangement of immunoglobulin genes, cells-cells at widely different stages of development- eliminates the need to elute the separated DNA restriction Tonegawa and Hozumi used various restriction endonucle- fragments from gel slices prior to analysis by hybridization ases to generate DNA fragments. The fragments were then with an immunoglobulin gene segment probe Figure 5-2 separated by size and analyzed for their ability to hybridize shows the detection of rearrangement at the k light-chain lo- with a radiolabeled mRNA probe. Two separate restriction by cor fragments produced by digestion of fragments from the embryonic DNA hybridized with the DNA from a clone of B-lineage cells with the pattern ob- mRNA, whereas only a single restriction fragment of the tained by digestion of non-B cells(e. g, sperm or liver cells) adult myeloma DNA hybridized with the same probe. Tone- The rearrangement of a V gene deletes an extensive section of gawa and Hozumi suggested that, during differentiation of germ-line DNA, thereby creating differences between re- lymphocytes from the embryonic state to the fully differenti- arranged and unrearranged Ig loci in the distribution and ted plasma-cell stage (represented in their system by the number of restriction sites. This results in the generation of Germ line Rearranged Deleted ∥[c →5-作口[c}3 Rearrangement Probe Probe Re digestion RE digestion Germ line Southern FIGURE 5.2 Experimental basis for diagnosis of rearrangement at are separated in the germ line. Consequently, fragments dependent an immunoglobulin locus. The number and size of restriction frag. on the presence of this segment for their generation are absent from ments generated by the treatment of DNA with a restriction enzyme the restriction-enzyme digest of DNA from the rearranged locus. Fur is determined by the sequence of the DNA. The digestion of re- thermore, rearranged DNA gives rise to novel fragments that are ab ranged DNA with a restriction enzyme(RE) yields a pattern of re- sent from digests of DNa in the germ-line configuration. This can be striction fragments that differ from those obtained by digestion of an useful because both B cells and non-B cells have two immunoglob unrearranged locus with the same RE. Typically, the fragments are an- lin loci. One of these is rearranged and the other is not. Consequently, alyzed by the technique of Southern blotting. In this example, a probe unless a genetic accident has resulted in the loss of the germ-line lo- that includes a j gene segment is used to identify RE digestion frag. cus, digestion of DNA from a myeloma or normal B-cell clone will ments that include all or portions of this segment. As shown, re. produce a pattern of restriction that includes all of those in a germ- arrangement results in the deletion of a segment of germ-line dna line digest plus any novel fragments that are generated from the and the loss of the restriction sites that it includes. It also results in change in DNA sequence that accompanies rearrangement. Note the joining of gene segments, in this case a V and a segment, that that only one of the several gene segements present is shownTonegawa’s Bombshell—Immunoglobulin Genes Rearrange In 1976, S. Tonegawa and N. Hozumi found the first direct evidence that separate genes encode the V and C regions of immunoglobulins and that the genes are rearranged in the course of B-cell differentiation. This work changed the field of immunology. In 1987, Tonegawa was awarded the Nobel Prize for this work. Selecting DNA from embryonic cells and adult myeloma cells—cells at widely different stages of development— Tonegawa and Hozumi used various restriction endonucle￾ases to generate DNA fragments. The fragments were then separated by size and analyzed for their ability to hybridize with a radiolabeled mRNA probe. Two separate restriction fragments from the embryonic DNA hybridized with the mRNA, whereas only a single restriction fragment of the adult myeloma DNA hybridized with the same probe. Tone￾gawa and Hozumi suggested that, during differentiation of lymphocytes from the embryonic state to the fully differenti￾ated plasma-cell stage (represented in their system by the myeloma cells), the V and C genes undergo rearrangement. In the embryo, the V and C genes are separated by a large DNA segment that contains a restriction-endonuclease site; during differentiation, the V and C genes are brought closer together and the intervening DNA sequence is eliminated. The pioneering experiments of Tonegawa and Hozumi employed a tedious and time-consuming procedure that has since been replaced by the much more powerful approach of Southern-blot analysis. This method, now universally used to investigate the rearrangement of immunoglobulin genes, eliminates the need to elute the separated DNA restriction fragments from gel slices prior to analysis by hybridization with an immunoglobulin gene segment probe. Figure 5-2 shows the detection of rearrangement at the light-chain lo￾cus by comparing the fragments produced by digestion of DNA from a clone of B-lineage cells with the pattern ob￾tained by digestion of non-B cells (e.g., sperm or liver cells). The rearrangement of a V gene deletes an extensive section of germ-line DNA, thereby creating differences between re￾arranged and unrearranged Ig loci in the distribution and number of restriction sites. This results in the generation of 108 PART II Generation of B-Cell and T-Cell Responses FIGURE 5-2 Experimental basis for diagnosis of rearrangement at an immunoglobulin locus. The number and size of restriction frag￾ments generated by the treatment of DNA with a restriction enzyme is determined by the sequence of the DNA.The digestion of re￾arranged DNA with a restriction enzyme (RE) yields a pattern of re￾striction fragments that differ from those obtained by digestion of an unrearranged locus with the same RE. Typically, the fragments are an￾alyzed by the technique of Southern blotting. In this example, a probe that includes a J gene segment is used to identify RE digestion frag￾ments that include all or portions of this segment. As shown, re￾arrangement results in the deletion of a segment of germ-line DNA and the loss of the restriction sites that it includes. It also results in the joining of gene segments, in this case a V and a J segment, that are separated in the germ line. Consequently, fragments dependent on the presence of this segment for their generation are absent from the restriction-enzyme digest of DNA from the rearranged locus. Fur￾thermore, rearranged DNA gives rise to novel fragments that are ab￾sent from digests of DNA in the germ-line configuration. This can be useful because both B cells and non-B cells have two immunoglobu￾lin loci. One of these is rearranged and the other is not. Consequently, unless a genetic accident has resulted in the loss of the germ-line lo￾cus, digestion of DNA from a myeloma or normal B-cell clone will produce a pattern of restriction that includes all of those in a germ￾line digest plus any novel fragments that are generated from the change in DNA sequence that accompanies rearrangement. Note that only one of the several J gene segements present is shown. 5′ 3′ Vn RE V2 RE J C V1 RE RE RE 5′ 3′ Vn RE V2 RE V1 RE RE RE Germ line Rearranged Germ line Rearranged Deleted Rearrangement J C Probe Probe RE digestion RE digestion Southern blot 8536d_ch05_105-136 8/22/02 2:46 PM Page 108 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e: